TGF-beta dynamically regulates the differentiation of smooth muscle cells, and has been postulated to maintain vessel wall structure. TGF-beta also appears to possess immunosuppressive properties which protect the vascular endothelium against local inflammation and damage. Moreover, TGF-beta may inhibit the proliferation and migration of smooth muscle cells after vascular injury.
TGF-beta is synthesized as a latent peptide (FIG. 1). Latent TGF-beta refers to any of several complexes that include the 25 kD TGF-beta dimer in association with the latency associated peptide (LAP) or any of several additional TGF-beta binding proteins (LTBPs). Latent TGF-beta has no biological activity, i.e., it does not bind to the TGF-beta receptors.
The 25 kD TGF-beta dimer is also found associated with matrix components or other plasma proteins (FIG. 1). TGF-beta that is associated with matrix components or other plasma proteins is termed mature TGF-beta. This association also prevents the binding of TGF-beta to the TGF-beta receptors.
In addition to latent and mature forms of TGF-beta, which cannot bind to the TGF-beta receptors and which possess no known biological activity, TGF-beta also exists in forms which are capable of binding to the TGF-beta receptors and which elicit biological effects (FIG. 1). These forms of TGF-beta are termed xe2x80x9cactive TGF-beta.xe2x80x9d One example of a form of active TGF-beta is the 25 kD TGF-beta dimer which is free from association with LAP/LTBPs, or matrix or plasma components. The process(es) by which the latent form of TGF-beta is converted to the active form is termed xe2x80x9cactivation.xe2x80x9d The process(es) by which the mature form of TGF-beta is converted to the active form is termed xe2x80x9crelease.xe2x80x9d
Decreased levels of TGF-beta have been implicated in the development of atherosclerosis. Atherosclerosis is a disease of the major arteries, typified by changes in the vessel wall architecture. At lesion-prone sites where the endothelium becomes damaged or dysfunctional, smooth muscle cells from the media of the vessel migrate into the intima. At these sites, leukocytes, and in particular, monocytes and macrophages invade the expanded intima. As the lesion develops, lipid from the circulation is deposited into the intima (reviewed in Ross, Nature, 362 801 (1993); Grainger et al. Biol. Rev. Camb. Philos. Soc., 70, 571 (1995)).
Agents which elevate TGF-beta activity, such as tamoxifen (TMX) (Grainger et al., Biochem. J., 294, 109 (1993)) and aspirin (Grainger et al., Nat. Med., 1, 74 (1995)), can exhibit cardioprotective effects. However, the positive cardioprotective effects of these agents may be counterindicated by their potential side effects. TMX can cause liver carcinogenicity in rats, has been correlated with an increased risk of endometrial cancer in women and may increase the risk of certain gut cancers. Aspirin may result in ulcerogenesis and increased bleeding.
Agents which elevate TGF-beta levels may also be useful to prevent or treat diseases or conditions including cancer, Marfan""s syndrome, Parkinson""s disease, fibrosis, Alzheimer""s disease, senile dementia, osteoporosis, diseases associated with inflammation, such as rheumatoid arthritis, multiple sclerosis and lupus erythematosus, and other auto-immune disorders. Such agents may also be useful to promote wound healing and to lower serum cholesterol levels.
Thus, there is a need for improved therapeutic methods and agents useful to maintain or elevate TGF-beta levels in mammals.
The present invention provides a method to maintain or elevate TGF-beta levels in a mammal, such as a human, in need of such therapy. The method comprises administering an effective amount of an aspirinate as defined herein. The method can also be carried out by administering an amount of a first therapeutic agent effective to elevate the level of latent TGF-beta and an amount of a second therapeutic agent effective to increase the level of TGF-beta which is capable of binding to the TGF-beta receptors, wherein said amounts are effective to maintain or elevate the level of TGF-beta in said mammal.
The invention also provides a method of preventing or treating a mammal, such as a human, having, or at risk of, a vascular indication which is associated with a TGF-beta deficiency. The method comprises the administration of an amount of an aspirinate that elevates the level of TGF-beta in said mammal so as to inhibit or reduce diminution in vessel lumen diameter. Preferably, the levels of active TGF-beta are elevated after administration of the aspirinate.
Preferred agents useful in the practice of the invention are copper aspirinates. Preferably, the effective amount of aspirinate inhibits lipid accumulation, increases plaque stability, decreases lesion formation or development, promotes lesion regression, or any combination thereof. Agents useful in the practice of the method include aspirinate salts such as copper salts of aspirinates, including copper aspirinate itself (copper 2-acetylsalicylate or copper 2-acetoxybenzoate), salicylate salts such as copper salts of salicylates, including copper salicylate (copper 2-hydroxybenzoate), or a compound of formula (I) (see below) including a pharmaceutically acceptable salt thereof, or a combination thereof.
An aspirinate useful in the present invention is a compound of formula (I): 
wherein
R1 is hydrogen, halo, nitro, cyano, hydroxy, CF3, xe2x80x94NRcRd, xe2x80x94C(xe2x95x90O)ORe, xe2x80x94C(xe2x95x90N)ORe xe2x80x94OC(xe2x95x90O)ORe, (C1-C6)alkyl or (C1-C6)alkoxy;
R2 is hydrogen or xe2x80x94XRa;
R3 is xe2x80x94C(xe2x95x90O)YRb, or xe2x80x94N(Rf)C(xe2x95x90O)Rgxe2x80x94;
R4 is (xe2x95x90O)n; or R4 is (C1-C6)alkyl, (C1-C6)alkanoyl or (C2-C6)alkanoyloxy;
R5 is hydrogen, xe2x80x94C(xe2x95x90O)ORh or xe2x80x94C(xe2x95x90O)SRh;
n is 0, 1 or 2;
X is oxygen, xe2x80x94N(Ri)xe2x80x94, or sulfur;
Y is oxygen or sulfur;
Ra is (C1-C6)alkanoyl, (C1-C6)alkyl, or hydrogen;
Rb is hydrogen or (C1-C3)alkyl;
Rc and Rd are each independently hydrogen, (C1-C4)alkyl, phenyl, C(xe2x95x90O)OH, C(xe2x95x90O)O(C1-C4)alkyl CH2C(xe2x95x90O)OH, CH2C(xe2x95x90O)O(C1-C4)alkyl, or (C1-C4)alkoxy; or Rc and Rd together with the nitrogen to which they are attached are a 3, 4, 5, or 6 membered heterocyclic ring; and
Re-Ri are independently hydrogen or (C1-C6)alkyl;
a pharmaceutically acceptable salt thereof; or a combination thereof;
provided that R2 and R3 are on adjacent positions of the ring to which they are attached, or are on the 2- and 5-positions of the ring; and further provided that when R2 is hydrogen; R3 is on the 2-or 5-position of the ring to which it is attached and R4 is (C1-C4)alkanoyloxy. Preferably, the compound of formula (I) is not 3-acetoxy-2-carboxythiophene.
Also provided is a method of preventing or treating a mammal having, or at risk of, a vascular indication by administering there to an amount of a first therapeutic agent and an amount of a second therapeutic agent which together are effective to elevate the level of TGF-beta, preferably the level of active TGF-beta, in said mammal. Preferably, the administration inhibits or reduces diminution in vessel lumen diameter. The inhibition or reduction in diminution in vessel lumen diameter preferentially occurs at a site in a vessel where the vascular indication is, or is likely to be, manifested. The invention thus provides for combination therapy, e.g., the administration of one agent that can elevate the level of latent TGF-beta, and another agent that can elevate the level of TGF-beta which is available to bind to, or is capable of binding to, the TGF-beta receptor. This combination therapy can yield a significantly greater cardiovascular efficacy than would be expected from the administration of either agent singly. The therapeutic agents can act in a synergistic, rather than in an additive, manner to elevate TGF-beta levels. The therapeutic agents can be administered simultaneously in a single dosage form simultaneously in individual doses, or sequentially.
A first therapeutic agent useful in this embodiment of the invention includes an aspirinate, e.g., a compound of formula (I). Another preferred first therapeutic agent comprises a compound of formula VI (see below). A preferred second therapeutic agent useful in this embodiment of the invention comprises at least one omega-3 fatty acid, which can be provided, e.g., by dosages of fish oil. Another preferred second therapeutic agent is selected from at least one compound of formula VI. Thus, a compound of formula VI may both elevate latent levels of TGF-beta and elevate the levels of TGF-beta which can bind to the TGF-beta receptors. Preferably, the combination of the therapeutic agents inhibits lipid accumulation, increases plaque stability, decreases lesion formation or development, promotes lesion regression, or any combination thereof.
A compound useful in the present invention is a compound of formula (VI): 
wherein
R6 is (C1-C6)alkyl, or aryl, optionally substituted by 1, 2, or 3 V;
R7 is phenyl, optionally substituted by 1, 2, or 3 V; or R7 is (C1-C12)alkyl, halo(C1-C12)alkyl, (C1-C6)cycloalkyl, (C1-C6)alkylcyclo(C1-C6)alkyl, (C1-C6)cycloalkenyl, or (C1-C6)alkyl(C1-C6)cycloalkenyl;
R8 is hydrogen or phenyl, optionally substituted at the 2-position with Rj, and optionally substituted by 1, 2, or 3 V;
R9 is hydrogen, nitro, halo, aryl, heteroaryl, aryl(C1-C3)alkyl, heteroaryl(C1-C3)alkyl, halo(C1-C12)alkyl, cyano(C1-C12)alkyl, (C1-C4)alkoxycarbonyl(C1-C6)alkyl, (C1-C12)alkyl, (C1-C6)cycloalkyl, (C1-C6)alkylcyclo(C1-C6)alkyl, (C1-C6)cycloalkenyl, or (C1-C6)alkyl(C1-C6)cycloalkenyl, wherein any aryl or heteroaryl may optionally be substituted by 1, 2, or 3, V; or
R9 and Rj together are xe2x80x94CH2CH2xe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94 xe2x80x94N(H)xe2x80x94, xe2x80x94N[(C1-C6)alkyl]xe2x80x94, xe2x80x94OCH2xe2x80x94, xe2x80x94OC[(C1-C6)alkyl]2xe2x80x94, or xe2x80x94CHxe2x95x90CHxe2x80x94;
xe2x80x94 is a single bond or is xe2x80x94C(B)(D)xe2x80x94, wherein B and D are each independently hydrogen, (C1-C6)alkyl, or halo;
V is OPO3H2, (C1-C6)alkyl, (C1-C6)alkoxy, mercapto, (C1-C4)alkylthio, halo, trifluoromethyl, pentafluoroethyl, nitro, N(Rn)(Ro), cyano, trifluoromethoxy, pentafluoroethoxy, benzoyl, hydroxy, xe2x80x94(CH2)0-4C(xe2x95x90O)(C1-C6)alkyl, xe2x80x94UC(xe2x95x90O)(C1-C6)alkyl, benzyl, xe2x80x94OSO2(CH2)0-4CH3, xe2x80x94U(CH2)1-4COORp, xe2x80x94(CH2)0-4COORp, xe2x80x94U(CH2)2-4ORp, xe2x80x94(CH2)0-4ORp, xe2x80x94U(CH2)1-4C(xe2x95x90O)Rk, xe2x80x94(CH2)0-4C(xe2x95x90O)Rk, xe2x80x94U(CH2)1-4Rk, xe2x80x94(CH2)0-4Rk, or xe2x80x94U(CH2)2-4OC(xe2x95x90O)Rp; wherein U is O, N(Rm), or S;
Z is xe2x80x94(CH2)1-3xe2x80x94, O, xe2x80x94OCH2xe2x80x94, xe2x80x94CH2Oxe2x80x94, xe2x80x94C(xe2x95x90O)Oxe2x80x94, xe2x80x94N(Rq)xe2x80x94, Cxe2x95x90O, or a covalent bond;
Rk is amino, optionally substituted with one or two (C1-C6)alkyl; or an N-heterocyclic ring optionally containing 1 or 2 additional N(R1), S, or nonperoxide O, wherein R1 is H(C1-C6)alkyl, phenyl, or benzyl;
Rn and Ro are independently hydrogen, (C1-C6)alkyl, phenyl, benzyl, or (C1-C6)alkanoyl; or Rn and Ro together with the nitrogen to which they are attached are a 3, 4, 5, or 6 membered heterocyclic ring;
Rp is H or (C1-C6)alkyl; and
Rm and Rq are independently hydrogen, (C1-C6)alkyl, phenyl, benzyl, or (C1-C6)alkanoyl;
the compound is MER25;
or a pharmaceutically acceptable salt thereof.
As described hereinbelow, the combination of aspirin plus an agent such as fish oil that increases the level of TGF-beta which is capable of binding to the TGF-beta receptors, results in a greater reduction in lesion formation in apoE knockout mice relative to aspirin or fish oil therapy alone. Surprisingly, the combination of aspirin and fish oil, which comprises a plurality of omega-3 fatty acids, exerts a markedly synergistic, rather than an additive, effect. Thus, a combination of an agent that elevates the level of latent TGF-beta, e.g., low doses of aspirin or an aspirinate, with an agent that increases the level of TGF-beta which can bind to its receptor, e.g., at least one omega-3 fatty acid, can be very effective in preventing or treating vascular disease. As used herein, xe2x80x9cat least onexe2x80x9d omega-3 fatty acid reflects the fact that one of skill in the art would recognize that natural sources of omega-3 fatty acids contain a plurality, about 1 to 30, preferably about 1 to 25, and more preferably about 2 to 20, of omega-3 fatty acids.
Another embodiment of the invention is a method for preventing atherosclerosis in a mammal at risk therefor, or treating atherosclerosis in a mammal, by administering to the mammal an amount of a first therapeutic agent and an amount of a second therapeutic agent effective to maintain or elevate the level of TGF-beta. The first therapeutic agent preferably increases the level of latent TGF-beta, e.g., is aspirin or an aspirinate, or a combination thereof, and the second therapeutic agent increases the level of TGF-beta which is capable of binding to the TGF-beta receptors. Thus, the agents of the invention are administered in a combined amount that prevents or inhibits diminution in vessel lumen diameter at, or near, a site or potential site of atherosclerotic lesion formation or development. A preferred first therapeutic agent comprises aspirin or an aspirinate. A preferred second therapeutic agent comprises at least one omega-3 fatty acid.
The invention also provides a method to inhibit diminution in mammalian vessel lumen diameter. The method comprises administering to a mammal in need of said therapy, an amount of a first therapeutic agent and an amount of a second therapeutic agent effective to maintain or elevate the level of TGF-beta, so as to inhibit or reduce vessel lumen diminution. The inhibition or reduction in diminution in vessel lumen diameter preferentially occurs at a site in a vessel where the diminution is or is likely to be manifested. The first therapeutic agent increases the level of latent TGF-beta, with the proviso that the first therapeutic agent is not aspirin. The first therapeutic agent is preferably an aspirinate. The second therapeutic agent increases the level of TGF-beta which is capable of binding to the TGF-beta receptors.
Also provided is a combination therapy to maintain or elevate TGF-beta levels in a mammal in need of such treatment. The method comprises the administration of an amount of a first therapeutic agent and a second therapeutic agent, wherein said amount is effective to maintain or elevate the level of TGF-beta. The first therapeutic agent increases the level of latent TGF-beta, while the second therapeutic agent increases the level of TGF-beta which is capable of binding to the TGF-beta receptors. A preferred first therapeutic agent comprises aspirin or an aspirinate, while a preferred second therapeutic agent comprises at least one omega-3 fatty acid.
The invention also provides a method to maintain or elevate TGF-beta levels in a mammal in need of such treatment. The method comprises the administration of an amount of an aspirinate effective to maintain or elevate the level of TGF-beta, preferably active TGF-beta, in said mammal.
The invention also provides a method of preventing or treating a mammal having, or preventing in a mammal at risk of, a condition which is associated with a TGF-beta deficiency. Also provided is a method to maintain TGF-beta levels in a mammal. The methods comprise the administration of one or more agents in an amount effective to elevate or maintain the level of TGF-beta in said mammal. The effective amount of the agent or agents may increase the level of latent TGF-beta or the level of TGF-beta which is capable of binding to the TGF-beta receptors. Agents useful to increase the level of latent TGF-beta include, but are not limited to, idoxifene, toremifene, raloxifene, droloxifene, ethynyl estradiol, diethylstibestrol, 1,25 dihydroxy-vitamin D3, retinoic acid and ligand pharmaceutical analogs thereof (Mukherjee et al. Nature, 1997, 386: 407-410), dexamethasone, progesterone, thyroid hormone analogues (e.g. sodium liothyronine and sodium levothyroxine), hexamethylene bisacetamide, 4-hydroxyquinazoline, coumarin and benzocaine.
Agents useful to increase the level of TGF-beta which is capable of binding to the TGF-beta receptors include agents that cause the release of TGF-beta from matrix components or plasma proteins, e.g., agents such as heparin sugar analogs and betaglycan proteoglycan chains, or cause the release of TGF-beta from lipoproprotein complexes, e.g., agents such as vitamin E, simvastatin, VLDL-lowering agents, Apo-AII-lowering agents, and ApoAI-stimulating agents. Other agents useful to increase the level of TGF-beta which is capable of binding to the TGF-beta receptors include agents that cause an increase in the conversion of the latent form of TGF-beta to the active form of TGF-beta, e.g., hydrocortisone, dexamethasone, compounds of formula VI, vitamin D3, retinoic acid, simvastatin and thrombospondin.
Also provided is a kit comprising packing material enclosing, separately packaged, at least one device adapted for the delivery of a unit dosage form of a therapeutic agent and at least one unit dosage form comprising an amount of at least one of the therapeutic agents of the invention effective to accomplish at least one of the therapeutic results described herein when administered locally or systemically, as well as instruction means for its use, in accord with the present methods. As used herein, a xe2x80x9cdevice adapted for deliveryxe2x80x9d of a therapeutic agent includes, but is not limited to, a catheter, a stent, a stet, a shunt, a synthetic graft, and the like.
Also provided is a kit comprising packing material enclosing, separately packaged, at least one device adapted for the delivery of a therapeutic agent to a site in the lumen of a mammalian vessel and at least one unit dosage form of a first therapeutic agent and one unit dosage form of a second therapeutic agent effective to accomplish at least one of the therapeutic results described herein when administered locally or systemically, as well as instruction means for its use, in accord with the present methods.
Further provided is a pharmaceutical composition comprising a) at least one aspirinate, and b) at least one omega-3 fatty acid, wherein components (a) and (b) are present in a combined amount effective to maintain or increase TGF-beta levels, preferably at or near a site, or potential site, of atherosclerotic lesion formation or development.
The invention also provides a pharmaceutical composition comprising (a) an amount of a first agent effective to elevate the level of latent TGF-beta; and (b) an amount of a second agent effective to increase the level of TGF-beta which is capable of binding to the TGF-beta receptors.
The invention also provides a pharmaceutical composition comprising a) an aspirinate, such as copper 2-acetylsalicylate or a compound of formula (I), and b) a compound of formula (VI), wherein components (a) and (b) are present in a combined amount effective to maintain or increase TGF-beta levels, preferably at or near a site, or potential site, of atherosclerotic lesion formation or development.
Also provided are novel compounds of formula (I), (II), (III), (IV), (V), (VI), (VII), or (VIII) or pharmaceutically acceptable salts thereof, and pharmaceutical compositions comprising a novel compound of formula (I), (II), (III), (IV), (V), (VI), (VII), or (VIII) as described herein or a pharmaceutically acceptable salt thereof, which are useful alone, or in combination, to elevate the level of TGF-beta in a mammal.
The invention also provides a therapeutic method. The method comprises identifying a patient exhibiting a decreased level of active TGF-beta and afflicted with a pathology associated with said decreased level. The patient so identified can be treated with an agent that elevates the levels of active TGF-beta so as to alleviate at least one of the symptoms of said pathology.
The invention also provides a method comprising determining endothelial cell activation in a mammal by detecting immunoglobulins that specifically bind to a TGF-xcex2 Type II receptor or a portion thereof.
The invention also provides a method comprising diagnosing or monitoring a disease characterized by endothelial cell activation (e.g. atherosclerosis) in a mammal by detecting immunoglobulins that specifically bind to a TGF-xcex2 Type II receptor or a portion thereof.
The invention also provides a method comprising detecting mammalian cells having TGF-xcex2 Type II receptors, by combining the cells with a capture moiety that binds TGF-xcex2 type II receptors or a portion thereof, forming a capture complex; and detecting or determining the amount of the capture complex.
The invention also provides a kit comprising packaging material containing: a) a capture moiety comprising the extracellular domain of the TGF-xcex2 Type II receptor; and b) a detection moiety capable of binding to an immunoglobulin. The invention also provides a kit comprising packaging material containing: a) a capture moiety that binds to the extracellular domain of the TGF-xcex2 Type II receptor; and b) a detection moiety capable of binding to an immunoglobulin.